Three‐dimensional structure of proteinase K at 0.15‐nm resolution

Abstract

The crystal and molecular structure of proteinase K was determined by X‐ray diffraction data to 0.15‐nm resolution. The enzyme belongs to the subtilisin family with an active‐site catalytic triad Asp39–His69–Ser224 but is a representative of a subgroup with a free Cys73 close to and ‘below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34–Cys123 and Cys178–Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel β‐sheet decorated by six α‐helices, three short antiparallel β‐sheets, 18 β‐turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+‐binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate‐recognition site (Ser132–Gly136), probably associated with a cis → trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the ‘oxyanion hole' and is displaced by the C=O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three‐stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132–Leu133–Gly134 on one side and Gly100–Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39–His69–Serr224 triad. These latter residues display low thermal motion corresponding to well‐defined geometry and are hardly accessible to solvent molecules. whereas the recognition‐site amino acids are more flexible and partially exposed to solvent. Copyright © 1988, Wiley Blackwell. All rights reserved